Transformation troubles

[mmustafa]

Hi All!!

I am struggling with trying to get my plasmid into competent cells. I am currently using the Invitrogen Gateway Directional TOPO kits and One Shot TOP 10 competent cells and have had no success with the transformation process. Any clues anyone?????

[Cat]

How about +ve control???

[mmustafa]

I used the control plasmid provided with the competent cells. No colonies there either!!

[Cat]

O.K., you have too many things that can go wrong - anything in the procedure to bad cells. The most common problems: 1. Heat shock: too long, wrong temp; 2. wrong antibiotic in selection media. If you are sure that you did not make mistakes in the procedure, then you can assume bad cells.

[mmustafa]

I have been heat shocking for 30 to 45 sec at 42C. That should be okay, right?

[Cat]

That should be fine. Check the procedure step by step and see if you are doing anything different:

http://www.invitrogen.com/content/sfs/m ... 10_man.pdf

[mmustafa]

Nope! Nothing's different!!!!

[Cat]

Then your problem is with competent cells.

[mmustafa]

I got new stock of competent cells and performed transformation using a pUC19 control plasmid and got really tiny colonies. What does that mean??

[Cat]

If there are many small colonies, then your transformation probably worked and size problem is likely due to too much antibiotic in the media. If there are only few small colonies, then they are likely to be escapees...

[h2so4hurts]

Haha, or he didn't let the plates grow long enough...

Also, try adding 4uL of your PCR product into to TOPO reaction, especially if your PCR product is weak. Maybe try gel purifying your PCR product before the TOPO reaction.

[mmustafa]

I got really nice strong single band (correct size) when I ran my PCR product on a gel. That's why I was confident that the transformation would go smoothly!!